1. Field of the Invention
The invention relates to a preparation containing antigens possessing immunological properties, notably antigenic properties, characteristic of viruses of various forms of hepatitis, such as B viral hepatitis or various other forms of hepatitis such as those which are known to be developed in certain patients following blood transfusions, for example hepatites called "non A" or "non B". The invention relates more particularly to preparations of this type, which are characterised by high purity, the absence of proteins of human origin and, when it concerns preparations having antigen properties similar to those of the HBs antigens, free of Dane particles. It also relates to a method for the production of these antigens.
2. Description of the Prior Art
It is known that certain of the specific antigen properties of the virus of hepatitis must be attributed to an antigen called "HBs Antigen" or "HBsAg", essentially formed by the envelope of the virus of viral hepatitis of the B type (HBV) or Dane particle. This antigen, which possesses vaccinating properties with respect to viral hepatitis of type B, is, at the present time, essentially obtained from human serum specimens. However, no other sources for the supply of HBsAg, are by now available by reason of the particular characteristics of the virus of B hepatitis (HBV). It seems only capable of infecting man, the chimpanzee and, perhaps, a small number of other primates. It has not been possible hitherto to propagate it in vitro in cell cultures. Certainly lines of hepatocarcinomas which synthesize HBsAg are known. The use in man of cancerous cells for the production of particles with a vaccinating character runs up against quite comprehensible objections. The use in preventive therapy of HBs antigens of human origin is not however devoid of serious risks. In fact, they arise generally from persons who have been exposed to the virus of B viral hepatitis, so that the presence of Dane particles, sometimes also highly infectious, in preparations of HBsAg antigens of seric origin cannot always be completely excluded, even in the case of extremely pure preparations. The contents of the even highly purified preparations of HBsAg antigen from the state of the art in serum proteins or other possibly antigenic components, capable of inducing in the treated subjects troublesome immunitary reactions, are not negligible, by reason of their very low initial content of HBsAg with respect to the proteins of the initial serum (for example, of the order of 50 .mu.g of HBsAg with respect to 80 mg of protein in a 1 ml of serum).
In spite of the difficulties encountered to have available sufficient amounts of virus, it has however been established that the genome of the virus of B hepatitis is formed from a partially single stranded circular DNA molecule, of which the longest strand includes of the order of 3200 nucleotides (SUMMERS J. and coll. (1975) Proc. Nat. Sci. U.S.A. 72, 4597-4601). At the most, it has been possible to localise the gene coding the portion of the protein of the HBsAg antigen, which is responsible for the immunological properties of the envelope of the virus of B hepatitis. The position of this gene, named "S gene" results notably from the diagrammatic map of the genome of the Dane particle, which is given in FIG. 1 of the drawings.
FIG. 1 comprises a diagrammatic map of the DNA of the genome of the Dane particle. This DNA comprises two strands b.sub.1 and b.sub.2, the shortest of them (b.sub.2) being normally devoid of the portion shown by a dashed line in the drawing.
It is known that this DNA only includes a single EcoRI site.
The arrow f.sub.1 gives the direction of the numbering of the nucleotides from which the longest strand b.sub.1 is composed, and the arrow f.sub.2 gives the direction of the transcription of the S gene by the cellular machinery of the cells invaded by the virus of B hepatitis.
The EcoRI site can hence be numbered 0, or 3182 (in the case of B hepatitis viruses belonging to serotype 3182) (NATURE, 1979, vol. 281, p. 646-650).
The inner concentric circle e.sub.1 gives the scale in numbers of nucleotides. This circle enables the positions of certain of the parts of this DNA to be specified. The numbers 3', 5' and 5', 3', at the lower part of the map indicate the terminal ends bearing the same numbers in conventional representations of the ends of the nucleic acid chains.
At the most, the coding gene for the part of the protein of the HBsAg antigen has been localised. The position of this gene, named "S gene", is shown diagrammatically by the arrow S in the Figure. The "S gene" is essentially borne by the fragment of the longest strand b.sub.1 situated between the nucleotide positions 155 and 833 of the diagrammatic map of FIG. 1 in the direction of the transcription of the S gene.
The shortest strand (b.sub.2) of the genome of the Dane particle can be "repaired" in vitro in the presence of precursor nucleotides and of a polymerase, for example by the technique of T. A. LANDERS and coll., J. VIROL., 23, 1977, p. 368-376. The genomes so-repaired of the viral hepatitis viruses (or any DNA capable of coding for the same aminoacid sequences) will be denoted below by the abbreviation DNA HBV.
It is therefore an object of the invention to provide processes such as have been specified above, which are applicable generally to the study and to the expression in eukaryotic cells of all or part of the whole genomes of the viruses responsible for the various viral hepatites, more particularly of DNA HBV. It is also an object to provide for the production of modified vectors enabling the practising of these methods.
Lastly, it is more particularly an object of the invention to provide for the production of preparations containing antigens having the same immunological specificity as HBsAg or of a similar antigen of high purity, as has been indicated above, from a system other than human serum, which system is both reproducible and stable (or having a certain number of particular characteristics which can be made the subject of constant surveillance).